Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Surface Plasmon Resonance of CaBP 107
Fig. 2. A typical sensorgram. The sharp rise in signal during the first couple of
seconds of the association phase is an effect of the bulk concentration change, as is the
sharp decrease in signal at the beginning of the dissociation phase.
8. Repeat from step 3 using the next calcium sample.
9. When the three calcium experiments have been performed, repeat steps 3–7 using
buffer only as a control. This sensorgram should look like Fig. 3.
10. Place the pump inlet tubing in the EDTA-buffer flask and initiate the flow system
with the new buffer.
11. Repeat between steps 3 and 9 for the three EDTA samples and a buffer control.
12. If calcium is absolutely essential for the interaction, the association and dissociation
phases of the calcium samples will look like the one in Fig. 2 and the
sensorgrams of the EDTA samples will look like Fig. 3 (see Note 10). If the
EDTA samples interact with the surface, but weaker than in the calcium case,
the association will be slower and/or the dissociation faster. See below for quantitative
measurement of kinetics.
3.4. Quantitative Kinetic Experiment
1. Use a calcium or EDTA buffer depending on what you want to measure. Do not
forget to initiate the flow system if you have changed the flow buffer. Set the flow
rate to 5 µL/min.
2. Make a few test runs to determine a good concentration interval (follow steps 3–7).
3. Make six samples with different analyte concentration within the appropriate
concentration range dissolved in the flow buffer. The dispersion between the
concentrations should be approximately a factor of 2 (e.g., 1, 2, 5, 10, 20, and
50 nM). Each sample should be 150 µL.
4. Run sensorgrams of the six samples and one control (follow steps 3–7). The
association phases of these sensorgrams will be used to evaluate the association
rate constant kon.